Ethanol Promotes The Progression Of HCC By Stimulating EMT Via Activating Wnt-β-catenin Signaling Pathyway

[Backgrounds]Hepatocellular Carcinoma (HCC) is a common highly malignant tumor with high rate of relapse and metastasis, especially in Asia. HCC ranks the second in mortality rate among all malignant tumors in China. In recent years, with the improvement of medical technology conditions, survival rates of tomor patients have improved, but the incidence and mortality of HCC remains high, the causes and the underlying mechanisms of HCC are still largely unknown. Numerous studies have shown that chronic stress, such as chronic inflammation, heavy metal pollution, and excessive drinking, is closely associated with tumor development. Ethanol is a known risk factor for HCC. However it is still unclear whether and how ethanol promotes the progression and metastasis of HCC.EMT (epithelial-mesenchymal transition) is a complex process in which cells undergo a switch from epithelial phenotype to mesenchymal phenotype. During tumor progression, EMT allows benign tumor cells to infiltrate surrounding tissue and metastasize to distant sites. Metastasis is responsible for as much as 90% of cancer-associated mortality. EMT is closely related to tumor metastasis. Studies have found that Wnt-β-catenin signaling pathway can regulate EMT. However, it is unreported whether Ethanol promotes the progression of HCC by stimulating Epithelial-to-Mesenchymal Transition via activating Wnt-β-catenin signaling pathway. Researches have reported that salinomycin, which was used as a drug targeted to kill cancer stem cells, is an inhibitor of Wnt-β-catenin signaling pathway, thus we assumed that whether salinomycin could reverse the ethanol-induced EMT through inhibition of Wnt-β-catenin signaling pathway. This study intends to explore whether chronic ethanol exposure promotes the progression of HCC by stimulating EMT via activating Wnt-β-catenin signaling pathway, and whether salinomycin could reverse the ethanol-induced EMT through inhibition of Wnt-β-catenin signaling pathway.[Methods]To explosure the effects and involved mechanisms of ethanol promotes the progression of HCC, we first established the HCC cell model with chronic ethanol treatment (0.2%). We detected the effects of ethanol on colony formation, soft agar colony formation, migration, apoptosis and tumorigenicity of HCC cells. We then established the HCC animal model with chronic ethanol consumption (2%) to detect the effects of ethanol on tumorigenicity of HCC cells in nude mice. Besides, we analysis the EMT related genes (E-cadherin, Vimentin, β-catenin, MMP-3, MMP-9) through Western Blotting, and analysis the Wnt-β-catenin related genes through Western Blotting, Immunofluorescent assay and Immunohistochemistry assay. Meanwhile, we treated the HCC cells with salinomycin, and analysis the Wnt-β-catenin related genes and EMT related genes through Western Blotting. Finally, we analysis the inhibition effects of salinomycin on ethanol induced EMT of HCC cells through cell biological behavior experiment in vitro and tumor formation ability of nude mice in vivo.[Results]In this research, we established the HCC cell model with chronic ethanol treatment (0.2%) and HCC animal model with chronic ethanol consumption (2%) successfully. We found that chronic ethanol exposure promotes the colony-formation ability, soft agar colony formation ability, and migration ability of HCC cells in vitro. Besides, chronic ethanol exposure inhibits cis-platinum induced apoptosis of HCC cells. In vivo, chronic ethanol exposure promotes the tumorigenicity of HCC cells in nude mice, while chronic ethanol consumption promotes the growth and metastases of HCC cells in nude mice. In the mechanism research, We found that Epithelial to mesenchymal transition (EMT) is involved in the progression of ethanol induced HCC, and Wnt-β-catenin signal pathway is activated by ethanol in HCC. Furthermore, we found that salinomycin reverses ETOH induced EMT via inhibition of the Wnt-β-catenin signaling pathway.[Conclusion]1. Chronic ethanol exposure promotes the progression and metastasis of HCC cells, and inhibits cis-platinum induced apoptosis of HCC cells.2. Chronic ethanol exposure promotes the tumorigenicity of HCC cells in nude mice.3. Wnt-β-catenin signal pathway is activated by ethanol in HCC.4. EMT is involved in the progression of ethanol induced HCC, while salinomycin reverses ETOH induced EMT via inhibition of the Wnt-β-catenin signaling pathway.